Mapping the Bacterial Chromosome Help
When Hfr and F– cultures are mixed, conjugation can be stopped at any desired time by subjecting the mixture to the shearing forces of a Waring blender, which break the conjugation bridge. The sample is diluted immediately and plated on selective media, incubated, and then scored for recombinants. In addition to the selected marker, an Hfr strain must also carry a distal auxotrophic or sensitivity marker that prevents the growth of Hfr cells on the selective medium and thereby allows only recombinant cells to appear. This technique is called counter-selection. Because of the polarity with which the Hfr chromosome is transferred, the time at which various genetic markers appear in the recipient indicates their linear organization in the donor chromosome. At a given temperature, the transfer of the first half of the Hfr chromosome proceeds at a relatively uniform rate. Therefore, the time of entry of different markers into a recipient (F–) cell is a function of the physical distance between them.
EXAMPLE 10.11 An Hfr strain carrying the prototrophic markers a+, b+, c+ is mixed with an F– strain carrying the auxotrophic alleles a, b, c. Conjugation was interrupted at 5-min intervals and plated on media that revealed the presence of recombinants.
The order of the genes in the Hfr donor strain is ori—b+ —c+ —a+; b is less than 5 time units from the origin (ori); c is less than 5 time units from b; a is less than 5 time units from c.
When conjugation is allowed to proceed normally, the amount of time before the cytoplasmic bridge ruptures is apparently random. The nearer a marker is to the origin (leading end of donor chromosome), the greater its chances of appearing as a recombinant in a recipient cell. Donor and recipient cells are mixed for about an hour in broth and then placed on selective media that allows growth of F recombinants only for a specific marker. Counterselection against Hfr must also be part of the experimental design. The counterselective marker should be located as distally as possible from the selected marker so that unselected recombinants will not be lost by its inclusion. The frequencies with which unselected markers appear in selected recombinants are inversely related to their distances from the selected marker, provided they lie distal to it. Proximal markers more than 3 time units apart exhibit approximately 50% recombination, indicating that the average number of exchanges between them is greater than 1. Just at the point where gross mapping by conjugation becomes ineffective, i.e., for markers less than 2 time units apart, recombination mapping becomes very effective, permitting estimation of distances between closely linked genes or between mutant sites within the same gene. Distances between genes can be expressed in three types of units: (1) time units, (2) recombination units, or (3) chemical units.
EXAMPLE 10.12 If 1 min of conjugation is equivalent to 20 recombination units in E. coli, and the entire chromosome is transferred in 100 min, then the total map length is 2000 recombination units. If 107 nucleotide pairs exist in the chromosome, then 1 recombination unit represents 107 / .2000 = 5000 nucleotide pairs.
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